X tremegene 9 transfection reagent

HeLa cells (epithelial cervix adenocarcinoma; A.T.C.C. no. CRL-1573) and CAL-62 cells (anaplastic carcinoma, 8305C DSMZ number ACC 448) were cultured and transfected as described previously [27 (link)]. Cells were harvested 72 h post siRNA transfection. siRNA-mediated knockdown was performed using Dharmafect 1 transfection reagent (Dharmacon) according to the instruction manual. Gene-specific siRNAs were purchased from Dharmacon and used at a final concentration of 50–100 nM to silence hNAA30. Two different siRNAs targeting hNAA30 were used to ensure that phenotypes were specific for hNAA30 depletion: sihNAA30-1 Dharmacon cat. number D-009961-01 and sihNAA30-2 cat. number D-009961-05. For ARFRP1 knockdown, a pool of four different siRNAs were used (Dharmacon cat. number L-019250-00-0010). Non-targeting siRNApool cat. number D-001810-10 was used as a negative control. Knockdown efficiency was inspected routinely by Western blotting for all experiments. Plasmid transfection was performed using Roche X-tremeGENE9 transfection reagent. In experiments where cells were subjected to both siRNA and plasmid transfection, 20 μM carbobenzoxy-VAD (O-methyl)-fluoromethylketone (z-VAD-fmk) pan-Caspase inhibitor (R&D Systems Europe Ltd.) was added to avoid cell death.

Cell culture material including media and supplements were from Biochrom (Berlin, Germany). Cells were cultured in a humidified atmosphere with 5% CO₂ at 37 °C. Monkey kidney (COS-7)and human embryonic kidney (HEK293) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% L-glutamine and 1% pyruvate (Biochrom).
Heterologous cells were transiently transfected using X-treme gene 9 transfection reagent (Roche, Mannheim, Germany) or FuGene6 (Promega, Mannheim, Germany) according to manufacturers´ protocols. For antibody staining, transfection of single constructs and co-localization studies with organelle markers, cells were grown on 13 mm glass coverslips and transfected with 1 μg of each construct. For endocytosis assay, HEK293 cells were transfected with 2 μg of each construct. Cells were incubated for 24 h after transfection before staining, fixation with 4% paraformaldehyde, or endocytosis assays with Alexa Fluor® 488 AcLDL.

5637 bladder cancer-derived cells were seeded in 6-well plates. At 70–80% confluency the cells were co-transfected with a hCas9 expression vector (a gift from George Church, Addgene plasmid #41815) [41 (link)] and two gRNA vectors (targeting the regions flanking the enhancer), using X-tremeGENE 9 transfection reagent (Roche) according to the manufacturer’s instructions. Three days after transfection, the cells were trypsinized and seeded at low densities in 10-cm dishes. The remaining cells were used for DNA isolation using a QIAamp DNA Mini Kit (QIAGEN) after which deletion-specific PCR was performed using SuperTaq DNA polymerase (ThermoFisher Scientific, Waltham, Massachusetts, USA). Deletion of the 2.2 kb E1 enhancer region was confirmed by agarose gel electrophoresis and Sanger sequencing of the PCR amplicons. The ratio between non-deleted and deleted cells was used to evaluate the efficiency of the CRISPR/Cas9 system (Supplementary Fig. A). After 2 weeks, single cell colonies were harvested and DNA was isolated using a NucleoSpin Tissue XS kit (Macherey-Nagel, Düren, Germany). Deletion-specific PCR was performed (as above) to identify and select E1-deleted clones (see Supplementary Fig. B).

For transfections, a full-length linear HBV DNA (nt 1820-1820) with sticky ends was used.
As previously described, linear HBV monomers were excised from the plasmids by restriction enzyme digestion with 5 U of BspQI (New England Biolabs, Beverly, MA, USA) at 50 °C. The 3.2 kb fragments were gel purified with PureLink Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions and the DNA was quantified spectrophotometrically [20 (link)].
For transfections, cells were seeded in 6 or 24 well plates and grown to 60–70% confluence. Transfections were carried out using X-tremeGene 9 transfection reagent (Roche, Mannheim, Germany), according to the manufacturer’s recommendations. Transfection with a linear full-length HBV genome derivative from the pCH-9/3091 POL minus plasmid was used as control for evaluating the amount of remaining input DNA. For trans-complementation assays, cells were co-transfected with sgtF1b X(-) or sgtF4 X(-) genomes and sgtF1b HBx or sgtF4 HBx expression plasmids, respectively. Cells were maintained at 37 °C in 5% CO₂ atmosphere. After 6 h incubation, medium was replaced, and cultures were incubated for 72 or 96 h. In order to eliminate HBV DNA input, cell culture medium was collected every 24 h, cells were washed six times with PBS, and fresh medium was added.

To generate Ba/F3 and NIH-3T3 cell lines with stable overexpression of ERBB4 variants, Phoenix-Ampho packaging cells were transfected with retroviral pBABEpuro-gateway constructs encoding ERBB4 variants or enhanced GFP (eGFP) using FuGENE 6 transfection reagent (Promega) according to manufacturer's instructions. The retroviral supernatants of Phoenix-Ampho cells harvested 24 and 48 hours after transfection were incubated on Ba/F3 and NIH-3T3 cells for 6 hours on 2 consecutive days in the presence of 0.8 µg/mL hexadimethrine bromide (Polybrene, Sigma-Aldrich). Cell pools with stable expression were selected with 2 µg/mL puromycin (Gibco) and then maintained in 1 µg/mL puromycin for Ba/F3 cells, or with 6 µg/mL puromycin and then maintained in 3 µg/mL puromycin for NIH-3T3 cells.
To generate BEAS-2B cell lines with stable overexpression of ERBB4 variants, Platinum-E packaging cells were transfected with retroviral pMSCV-PGK-Puro-IRES-GFP constructs encoding ERBB4 variants or an empty vector using X-tremeGENE 9 transfection reagent (Roche) according to manufacturer's instructions. The retroviral supernatants of Platinum-E cells were harvested 48 hours after transfection and incubated on BEAS-2B cells for 72 hours. Transduced BEAS-2B cells were cultured in the presence of 1 µg/mL puromycin to select and maintain cell lines with stable expression.